Type 1 diabetes (T1D) results from a complex cascade of events that breaks immune tolerance and culminates in the destruction of islet cells. B lymphocytes (B cells) play a critical role in disease. This project focuses on the role ofa central B cell signaling protein, Bruton's tyrosine kinase (BTK). We have defined the role of BTK in supporting autoreactive B cells that present antigen to autoreactive T cells, and shown that BTK can be targeted to protect against development of T1D. We now extend those findings to mucosal immunity, and have discovered that BTK-deficient nonobese diabetic (NOD) mice have greatly reduced numbers of Peyer's Patch B cells, important for production of IgA. Accordingly, IgA is also reduced. We therefore tested the hypothesis that IgA in gut mucosa selects the resident microbes. Genetic sequencing of intestinal microbiota showed that Btk-/-/NOD mice have strikingly altered microbial populations compared with wild type NOD mice. Furthermore, exposure to different microbes in the neonatal period abrogated disease protection in Btk-/-/NOD mice. The hypothesis underlying this proposal is that B cell signaling via BTK supports production of IgA, which shapes a microbiome that promotes T1D in genetically susceptible hosts. To understand BTK contributions to the microbiome in relationship to T1D, we propose to: 1) determine the mechanisms by which BTK regulates the microbiome, by using cell-specific targeted deletion to define the effects on gut immune structures including Peyer's Patches. The impact of BTK actions on IgA production and gut immune responses will be investigated and effects of BTK inhibitors on mucosal immunity assessed, 2) determine the kinetics of BTK-targeting in modifying the microbiome. The developmental stages from neonate to adult when the microbiome is sensitive to the functions of BTK will be identified. Timed excision of BTK or drug administration will assess the potential to halt the progression of T1D in the NOD model and determine if microbiome modification is required for a successful outcome. Experiments will also determine how reconstitution with PP B cells affects microbial modification and disease outcome, and 3) determine disease contributions of the microbiome that is supported by BTK. Btk-/-/NOD mice will be rederived into a germ-free facility to separate autoimmune disease effects of BTK-signaling from that of the microbiome. Fecal transfer will be used to directly test microbiome contributions to T1D. This project has direct clinical importance in understanding how BCR-signaling supports the selection of commensal flora that influences the outcome of T1D as a necessary step in developing therapeutic interventions.